RESUMO
Sustained exposure to nicotine is well known to increase the cell surface density of α4ß2* neuronal nicotinic receptors both in vivo and in vitro, but the cellular mechanisms mediating this effect are equivocal. Using a pharmacological approach to investigate the effects of nicotine on receptor subunit expression and phosphorylation in SH-EP1 cells expressing human α4 and ß2 nicotinic receptor subunits, we have demonstrated that incubation with nicotine for 24 h increased the expression of immature and mature forms of both α4 and ß2 subunits in a concentration-dependent manner, and that inhibition of protein kinase C (PKC), but not cAMP-dependent protein kinase (PKA) inhibited the nicotine-induced increased expression of subunits. Incubation of cells with nicotine for 24 h also increased the phosphorylation of immature forms of α4 subunits similar to that induced by activation of either PKC or PKA. When cells were preincubated with nicotine, the PKC-mediated increased phosphorylation was inhibited; the PKA-mediated phosphorylation was unaltered. The phosphopeptide maps for immature α4 subunits following nicotine exposure or PKC activation were identical, and phosphoamino acid analyses indicated phosphorylation on serine residues only. Results indicate that nicotine-induced up regulation of α4ß2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, promoting subunit assembly and receptor maturation. Because up regulation of these receptors has been implicated to mediate tolerance, locomotor sensitization and addiction to nicotine, results identify a potential new target for modulating the effects of nicotine on the brain.
Assuntos
Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Proteína Quinase C/metabolismo , Receptores Nicotínicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Análise de Variância , Autorradiografia/métodos , Linhagem Celular Transformada , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Dibutirato de 12,13-Forbol/farmacologia , Isótopos de Fósforo/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Nicotínicos/genética , TransfecçãoRESUMO
Neuronal nicotinic receptor alpha4 subunits associated with nicotinic alpha4beta2 receptors are phosphorylated by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human alpha4 and/or beta2 subunits were incubated with (32)Pi, and PKA or PKC was activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing alpha4beta2 receptors or only alpha4 subunits were used to identify free alpha4 subunits, and alpha4 subunits present in immature alpha4beta2 complexes and mature alpha4beta2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of alpha4 subunits associated with mature pentamers was three times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free alpha4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature alpha4beta2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated beta2 subunits associated with mature alpha4beta2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation alpha4beta2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of human alpha4beta2 receptors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase C/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Análise de Variância , Anticorpos/farmacologia , Autorradiografia , Biotinilação , Linhagem Celular Transformada , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional/métodos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Imunoprecipitação , Indóis/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Modelos Biológicos , Técnicas de Patch-Clamp , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Mapeamento de Interação de Proteínas , Receptores Nicotínicos/genética , Receptores Nicotínicos/imunologia , Serina/metabolismo , Fatores de Tempo , Transfecção/métodosRESUMO
Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.
Assuntos
Oxirredutases/química , Rhodobacter sphaeroides/enzimologia , Técnicas Bacteriológicas , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Coenzimas/química , Deutério/química , Dietil Pirocarbonato/química , Ativação Enzimática , Nucleotídeos de Guanina/química , Cinética , Metaloproteínas/química , Cofatores de Molibdênio , NADP/química , Oxirredutases/antagonistas & inibidores , Oxirredutases/isolamento & purificação , Cianeto de Potássio/química , Pteridinas/química , Pterinas/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rhodobacter sphaeroides/crescimento & desenvolvimento , Especificidade por Substrato , Tungstênio/análiseRESUMO
Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin, an important step in oxidized vitamin salvaging. In addition to BSO, the enzyme also catalyzes the reduction of a variety of other substrates, including methionine sulfoxide, with decreased efficiencies, suggesting a potential role as a general cell protector against oxidative damage. Recombinant BSOR, expressed as a glutathione S-transferase fusion protein, contains the molybdopterin guanine dinucleotide cofactor (MGD) as its sole prosthetic group, which is required for the reduction of BSO by either NADPH or reduced methyl viologen. Comparison of the amino acid sequences of BSOR and the closely related MGD-containing enzyme, dimethyl sulfoxide reductase, has indicated a number of conserved residues, including an active site serine residue, serine 121, which has been potentially identified as the fifth coordinating ligand of Mo in BSOR. Site-directed mutagenesis has been used to replace serine 121 with cysteine, threonine, or alanine residues in the BSOR sequence to asses the role of this residue in catalysis and/or Mo coordination. All three BSOR mutant proteins were expressed, purified to homogeneity, and demonstrated to contain both MGD by fluorescence spectroscopy and Mo by inductively coupled plasma mass spectrometry, similar to wild-type enzyme. However, all three mutant proteins were devoid of BSOR activity using either NADPH or reduced methyl viologen as the electron donor. These results strongly suggest that serine 121 in BSOR is essential for catalysis but is not essential for either Mo coordination or MGD binding.
Assuntos
Coenzimas , Proteínas Ferro-Enxofre , Oxirredutases/metabolismo , Serina/química , Alanina/química , Sequência de Aminoácidos , Aminoácidos/química , Sítios de Ligação , Biotina/análise , Catálise , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Cisteína/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fator Xa/metabolismo , Glutationa Transferase/metabolismo , Nucleotídeos de Guanina/química , Ligantes , Espectrometria de Massas , Metaloproteínas/metabolismo , Dados de Sequência Molecular , Cofatores de Molibdênio , Mutagênese Sítio-Dirigida , NADP/metabolismo , Estresse Oxidativo , Oxirredutases/fisiologia , Oxigênio/metabolismo , Paraquat/metabolismo , Pteridinas/metabolismo , Pterinas/química , Proteínas Recombinantes de Fusão/metabolismo , Rhodobacter sphaeroides/enzimologia , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Treonina/química , Fatores de TempoRESUMO
Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 micromol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 microM for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.
Assuntos
Nucleotídeos de Guanina/metabolismo , Oxirredutases/metabolismo , Pterinas/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Nucleotídeos de Guanina/química , Cinética , NADP/metabolismo , Oxirredutases/química , Oxirredutases/isolamento & purificação , Pterinas/química , Proteínas Recombinantes de Fusão/química , Rhodobacter sphaeroides , Espectrofotometria UltravioletaRESUMO
The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the protein's composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.
Assuntos
Proteínas Ferro-Enxofre , Oxirredutases/química , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R. sphaeroides BSO reductase which has been sequenced. The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop. The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin. When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.